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Nanomaterials 2019, 9, 1042 4 of 20 2.6.1. TEM A carbon-coated formvar film was used to prepare nanoparticle samples for TEM analysis. The samples deposited on formvar film were air dried and placed on a copper grid for imaging under a JEOL JEM-2100F transmission electron microscope (Akishima, Tokyo, Japan) at 200 kV. 2.6.2. DLS and Zeta Potential Measurements A Nano ZS Zetasizer (Malvern Panalytical, Malvern, Worcestershire, UK) was used for measuring the size distribution of the nanoparticle suspensions by DLS and for calculating zeta potential to evaluate the stability of the nanoparticle suspensions. For the analysis of DLS and zeta potential, 3 mL of the nanoparticle suspension was used. 2.6.3. FTIR Spectroscopy FTIR spectroscopy was performed using a JASCO-FT/IR-6800 spectrophotometer (Hachioji, Tokyo, Japan) over the wavenumber range of 4000–800 cm−1. The lyophilized samples were mixed with potassium bromide (KBr) to form pellets for analysis. 2.6.4. EPMA A JEOL JXA-8530F electron probe micro-analyser (Akishima, Tokyo, Japan) was used for wavelength dispersive spectroscopy (WDS) for composition analysis. WDS measurements are established using Bragg’s law and require the use of multiple crystals as monochromators. The samples were dropped on a coverslip and air dried. Before analysis, the samples were covered with carbon and mounted on a copper grid. 2.7. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) MIC and MBC of AgNPs were determined against Gram-positive and Gram-negative microorganisms, namely Staphylococcus epidermidis NBRC100911 (S. epidermidis) and Escherichia coli K 12 (E. coli), respectively. The broth microdilution method, performed in a 96-well microplate, was employed with some modifications [28]. Nutrient broth was used to cultivate S. epidermidis, whereas LB broth was used for E. coli. Both cultivation media were procured from Difco Laboratories Inc., Sparks, MD, USA. AgNPs were tested for concentrations of 0.5–256 μg/mL. Streptomycin was used at the same concentrations as the positive control. Microbial cells were also exposed to the pigment concentration of 1–512 μg/mL to detect any antimicrobial activity. In brief, three to five morphologically-alike colonies grown at 37 ◦C for 24 h on agar plates were picked up and transferred to sterile fresh broth. This suspension was vortexed briefly to ensure uniform mixing, and the suspension turbidity was adjusted to 0.5 McFarland standard (OD625 0.08–0.13). The bacterial suspension was diluted 100 times before inoculating with the test/control substance in 1:1 ratio, resulting in a final concentration of 5 × 105 cells/mL. The final volume of the test solution in the 96-well plate was 100 μL, and the last two rows were employed as growth control and sterility control, respectively. Growth control was used to observe the growth of organisms without AgNPs or streptomycin, whereas sterility control was employed to ensure that there was no contamination. The 96-well plates were then incubated at 37 ◦C for 24 h. The lowest concentration which exhibited no visible growth compared to the control wells was considered the MIC value. To determine the MBC, the suspensions with concentrations higher than the MIC values were plated to check for cell survival, and the least concentration displaying no cell survival was deemed to be MBC. All the experiments were performed in triplicates. 2.8. Cell Death Kinetics Study S. epidermidis was used to study the cell death kinetics. Initial inoculum containing 1 × 106 cells/mL was exposed to the MBC dosages of AgNPs and streptomycin in a 96-well platePDF Image | Biosynthesis of Silver Nanoparticles Talaromyces purpurogenus
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